Eukaryotic protein translation initiation factor 4D (eIF-4D) contains one residue of hypusine and appears to be the only cellular protein with this one unique amino acid. Hypusine is produced post-translationally by transfer of the butylamine portion of the polyamine spermidine to a lysine residue in the eIF-4D precursor and subsequent hydroxylation. These findings reveal a novel cellular metabolic pathway. Comparison of activities of mature eIF-4D, eIF-4D precursors that contain unmodified lysine in place of hypusine and the deoxyhypusine containing eIF-4D prepared by in vitro modification of cloned eIF-4D precursor by deoxyhypusine synthase, in methionyl-puromycin synthesis indicates that deoxyhypusine and hypusine are essential for the activity of eIF-4D in this model protein synthesis initiation system. Studies are underway to relate the structure of hypusine to the physiological function of eIF-4D and to its mode of action in eukaryotic protein synthesis.